The problem of the entry, into living cells, of various substances having pharmacological properties, and of their access to the various intracellular compartments, in particular the cytoplasmic compartment and the nuclear compartment, is of great importance both for research and for therapeutic use.
A limited number of means for introducing substances such as polypeptides and oligonucleotides into the intracellular compartments is currently known. Among the various techniques currently proposed, there may be mentioned:
1. The transfection of genes, and derived techniques which make it possible to enhance in vivo or in vitro the transfection rates, such as precipitation with calcium phosphate, the use of cationic lipids, electroporation, trituration (scrape loading), the use of viral vectors, and the like.
2. The binding to cellular membrane receptors; these receptors are subsequently endocytozed, and release the bound molecules into the cytoplasmic compartment. In this category, there may be mentioned the folate receptor, the diphtheria toxin or transcription factors such as the TAT protein of the HIV retrovirus. The mechanism of transport involving these receptors is still poorly known, but requires in all cases an endocytosis stage.
3. The homeodomain-type peptides. Previous work carried out by the team of inventors on the homeodomain of the transcription factor Antennapedia (AntpHD) have made it possible to show that the homeodomain peptides cross the plasma membranes by an energy-independent process which is therefore distinct from endocytosis. The 3rd helix of the homeodomain peptide has the same properties [JOLIOT et al., Proc. Natl. Acad. Sci., USA, 88, p. 1864-1868 (1991); DEROSSI et al., J. Biol. Chem. 269, 14, p. 10444-10450, (1994)].
These properties have been used to internalize, in cells, polypeptides and oligonucleotides linked to the homeodomain or to helix 3, [PEREZ et al., J. Cell. Science, 102, p. 712-722, (1992)] by genetic fusion or biochemical bonding. This entry is quantitative, and the vector and its load are found in 100% of the cells; in addition, the internalization is independent of the relevant cell type.
The smallest fragment of the homeodomain capable of crossing the membranes and of serving as vector for other peptides or for oligonucleotides is a peptide of 16 amino acids, corresponding to helix 3. This peptide, which comprises amino acids 43 to 58 of the homeodomain, is named below. By way of example, the sequence of the peptide 43-58 of the homeodomain Antp is the following:
Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Met-Lys-Trp-Lys-Lys PA1 the peptide whose sequence is represented in the sequence listing in the annex under the number SEQ ID NO:1; PA1 the peptides in which X.sub.3 and X.sub.5 each represent a valine residue. PA1 intracellular translocation does not require a specific receptor, and can therefore affect all cellular types; PA1 the alpha-helix structure does not play a part in intracellular translocation, (but undoubtedly plays a role in nuclear addressing); PA1 the amphiphilic properties of the peptide, as well as the presence of a Trp residue appear, on the other hand, to be important for translocation.
This sequence is represented in the sequence listing in the annex under the number SEQ ID NO:1.
The mechanism by which this peptide could penetrate into living cells has been the subject of various studies, and it was assumed until now that its alpha-helix structure was essential for the internalization. The team of inventors has previously shown that certain substitutions or deletions in the peptide sequence, which modified the structure of the peptide, interfered with the activity of the said peptide. For example, a peptide in which the 2 Trp's (48 and 56) are replaced by two Phe's, or a peptide comprising amino acids 41 to 55 of the homeodomain are not internalized (DEROSSI et al., 1994, publication cited above). Another team [BRUGIDOU et al., Biophys. Biochem. Res. Com., 214:2, pp 685-693, (1995)] observed the internalization of peptides constituting structural analogues of the peptide 43-58; for that, they constructed, from a peptide 43-58 (differing from the peptide 43-58 of the homeodomain Antp in that the 2 isoleucines at positions 45 and 47 are replaced by valines), variants of the retro-inverse type. The retro-inverse variants, which make it possible to mimic the three-dimensional structure of natural peptides, consist of amino acids of the D series (instead of amino acids of the L series in the natural peptides) linked according to a sequence which is the reverse of that of the peptide to be reproduced.
The inventors have now sought to define the minimum characteristics of the amino acid sequences capable of serving as vector for the internalization and addressing of polypeptides and oligonucleotides, and have, for this purpose, synthesized several peptides by specifically modifying certain residues.